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Evolution, 56(5), 2002, pp. 1074–1079

A GENETIC CORRELATION BETWEEN AGE AT PUPATION AND MELANIZATION IMMUNE RESPONSE OF THE YELLOW FEVER MOSQUITO AEDES AEGYPTI 1 Laboratoire

JACOB C. KOELLA1,2 AND CHRISTOPHE BOE¨TE1 de Parasitologie Evolutive, CC237, Centre National de Recherche Scientifique, Unite´ Mixte de Recherche 7103, Universite´ P. et M. Curie, 7 quai St. Bernard, 75252 Paris, France 2 E-mail: [email protected]

Abstract. To investigate the evolutionary cost of an immune response, we selected six lines of the mosquito Aedes aegypti for earlier or later pupation and measured the extent to which this selection procedure changed the mosquito’s ability to encapsulate and melanize a negatively charged Sephadex bead. After 10 generations of selection, the age at pupation in the two selection regimes differed by about 0.7 days, accompanied by an increase of wing length of the mosquitoes selected for late pupation. Among the mosquitoes that had been selected for early pupation, only 6% had strongly or completely melanized the bead, while among the individuals that had been selected for late pupation, 32% had melanized the bead. Thus, our results suggest a genetic correlation between age at pupation and immunocompetence. As a consequence, mosquitoes that respond to increased intense parasite pressure with more effective immunity are predicted to pay for the increased defense with slower development. Key words.

Aedes aegypti, age at pupation, cost of immunity, development time, immunocompetence. Received September 25, 2001.

How much should an organism invest in its ability to mount an immune response against parasites? If immunocompetence is limited by the availability of resources, investing them in immune defense will reduce the resources available to other traits associated with the organism’s reproductive success, in particular to important life-history traits such as fecundity and age at maturity. Thus, evolutionary pressure will favor those individuals that allocate their resources to life-history traits and immunocompetence in a way that maximizes reproductive success. In other words, evolutionary pressure will lead to an investment of resources that balances the benefits and costs of immunity. The benefits of an immune system are clear. Parasites by definition reduce the reproductive success of animals, and an effective immune system will help to reduce their negative impact. There is also increasing evidence for a cost of immunity. Thus, inoculating nonpathogenic antigens can decrease the rate of growth of domestic mammals (Wagland et al. 1984; Spurlock et al. 1997) and birds (Klasing et al. 1987). In natural populations, female pied flycatchers whose immune system has been activated with nonpathogenic antigens decrease their feeding efforts and have lower reproductive success than control birds who have not been manipulated (Ilmonen et al. 2000). Similar patterns have been observed in other species of birds (Norris and Evans 2000), although not all species follow this common pattern (Williams et al. 1999). Similarly, forcing birds to increase their reproductive effort reduces the level of specific immune responses (Nordling et al. 1998). The same kinds of costs are found in insects. In bumblebees, increased foraging effort compromizes the encapsulation immune response (Ko¨nig and Schmid-Hempel 1995) and experimental activation of the immune system reduces life span (Moret and Schmid-Hempel 2000). In damselflies, increased reproductive effort decreases immunocompetence (Siva-Jothy et al. 1998). In Drosophila, the immune response

Accepted February 12, 2002.

to parasitoids reduces the resistance to starvation (Hoang 2001) and, in males, increased sexual activity reduces the rate at which bacteria are cleared by the immune system (McKean and Nunney 2001). Indirect evidence for a cost of the immune response is believed to be found in the common trade-off between investing in life-history traits and the risk of parasitism (Møller 1997; Sheldon and Verhulst 1996). This association may reflect a trade-off between life history and immunocompetence. While these and related studies give relatively clear evidence of a physiological cost of immunity, it is striking that very few studies have considered genetic correlations between immunity and life-history or other traits. In notable exceptions, artificial selection of a snail for increased resistance against its schistosome parasites led to lower fecundity (Webster and Woolhouse 1999), and selection of Drosophila larva for more efficient encapsulation of parasitoid eggs led to low competitive ability (Kraaijeveld and Godfray 1997). Yet, genetic correlations are of crucial importance for evolutionary change (Bell and Koufopanou 1986). In other words, to understand the evolutionary consequences of a cost of immunity, we must not only measure the phenotypic association between immunocompetence and reproductive success, but also the genetic correlation between the traits. In this study, we measured the correlated response of one aspect of a mosquito’s immunocompetence (its encapsulation and melanization responses) to selection on its age at pupation. The mosquito’s melanization response is known to be determined by its genotype (Gorman et al. 1996, 1997; Gorman and Paskewitz 1997), the conditions it has experienced during its development (Suwanchaichinda and Paskewitz 1998), and its reproductive status (Chun et al. 1995). The last suggests a trade-off of immunity with reproduction, whereas the genetic basis allows the possibility of a genetic correlation. Our emphasis is on the mosquito’s age at pupation (i.e., its developmental rate) because this trait is one of the most important in determining fitness (Stearns 1992).

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MATERIALS

AND

METHODS

Our experiment was done with the yellow fever mosquito Aedes aegypti, a species that is ubiquitous in the tropics and subtropics. As a vector of filarial worms and of yellow fever, dengue, and other viruses, it has been the subject of extensive investigations (Christophers 1960). We used a colony recently derived from a natural population in Senegal and maintained at several hundred females in each generation, so that its genetic diversity could be maintained at a high level. The experiment consisted of two parts: we first selected three lines of mosquitoes for early and three lines for late pupation; we then tested the immunocompetence of the selected lines. Selection Procedure In each generation we reared 200 mosquitoes per selection line in a climate chamber maintained at 288C (6 0.58C) and 80% (6 5%) relative humidity with a 12:12 L:D cycle. We hatched larvae synchronously by flooding eggs under reduced pressure for 30 min, added the larvae to 0.6 L of demineralized water in 25 3 12 3 4-cm plastic pans, and fed them with a standardized amount of the fish food Tetramin (Tetra Werke, Mille, Germany, day 1: 0.04 mg per larva; day 2: 0.06 mg; day 3: 0.12 mg; day 4: 0.24 mg; even-numbered days 6 onward: 0.48 mg). We used a divergent selection procedure to select for early or late pupation (thus eliminating the need for unselected controls; Falconer 1989). Within each selection line, we allowed only the 50 females that pupated earliest (or latest) and, to increase selection pressure, the 15 to 20 males that pupated earliest (or latest) to contribute to the following generation. Although the biased sex ratio decreases the effective population size, previous experiments suggest that these numbers would give a rapid response to selection (J. Koella, unpubl. data). Selection was continued for nine generations, and we used the offspring of the subsequent generation to measure immunocompetence. Immunocompetence To estimate immunocompetence, we reared 60 mosquitoes of each selection line individually in the wells of cellular culture polystyrene microplates (Iwaki Glass Co., Tokyo, Japan). Larvae were given the standard amount of food (see above). Individuals from each selection line that had pupated on a given day were put together into 0.3-L cups and were allowed to emerge. Adults were fed with a 0.1% glucose solution on an impregnated cotton that was changed every day. Females were tested for immunocompetence 4 days after emergence and within 1 h of having been blood-fed. Mosquitoes that had not obtained a full blood-meal were discarded. We decided to neglect the males because it would not be possible to separate the effect of sex on the immune function from the effect of the blood-meal. We stimulated the melanization response with negatively charged CM C-25 Sephadex beads. These beads are often used as a model system (Chun et al. 1995; Gorman and Paskewitz 1997; Gorman et al. 1997), as the immune response against them appears to share a genetic basis with the response against malaria parasites (Gorman et al. 1996). The

beads ranged 40–120 mm in diameter; the smallest ones were selected for inoculation by visual inspection. They were rehydrated in saline solution consisting of 1.3 mM NaCl, 0.5 mM KCl, 0.2 mM CaCl2, and 0.001% methyl green (pH 6.8; Paskewitz and Riehle 1994). We immobilized mosquitoes by chilling them briefly at 48C and inoculated one bead together with at most 0.3 ml of saline into the thorax. Inoculated mosquitoes were maintained in plastic cups humidified with soaked filter paper. After 48 h, we dissected the mosquitoes that were capable of moving (about 80% of the inoculated mosquitoes) and scored bead melanization according to three categories: (1) no visible melanization; (2) patchy melanization (i.e., leaving unmelanized areas on the bead); or (3) complete melanization of the bead. The selection line of the mosquito was not known at the time of dissection. The mortality due to inoculation was slightly lower in the lines selected for early pupation (18.6%) than in those selected for late pupation (21.1%), but the difference was not statistically significant (t 5 1.38, df 5 4, P 5 0.24). Body Size We measured the mean length of the two wings from the distal end of the alula to the tip of vein R3. Statistical Analysis The differences of age at pupation and wing length among selection lines were analyzed with a nested analysis of variance (ANOVA) that included the factors selection regime and line within selection regime. The latter was considered a random factor. We evaluated the correlated response of the melanization response to selection by analyzing the difference in melanization among selection lines with a nested ordinal logistic regression. This analysis estimates the probability that a given mosquito had one of the three levels of melanization response as a function of its selection regime and its line within selection regime, where the component of the difference explained by selection regime shows the correlated response to selection. The correlated response of body size was analyzed with a nested ANOVA of wing length as a function of selection regime and the random factor line within selection regime. For this analysis, we used only the mosquitoes that could be analyzed for their melanization response. All analyses were done with JMP version 4.0.4 (Available via http://www.jmpdiscovery.com). RESULTS Before selection, there was no difference of age at pupation between the selection regimes, but there was significant variation among selection lines within selection regimes, even though we had randomly chosen larvae to initiate the selection lines (Table 1). Although only the lines selected for rapid development responded to selection, the difference between slow and rapid lines rapidly increased up to about 0.7 days after five generations; between the fifth and ninth generation, selection had no additional effect (Fig. 1). After nine generations of selection, the three lines of mosquitoes selected for rapid development pupated on average after 6.1, 6.4, and 6.6 days, whereas the mosquitoes for slow development pu-

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TABLE 1. Analysis of variance of the response of age at pupation to selection for early or late pupation. Selection line is considered a random factor. Nine generations of selection

Before selection Source

Selection regime Line (selection) Error

Sum of squares

df

P

0.27 43.93 3185.47

1 4 1131

0.771 0.004

Sum of squares

df

P

31.07 5.91 884.40

1 4 471

0.015 0.534

pated after 6.9, 7.1, and 7.2 days. There was no statistically significant difference among selection lines within selection regime (Table 1). Of the approximately 180 females that had been reared in the 10th generation (half of the 6 3 60 5 360 larvae), 82 survived larval development, took an adequate blood-meal, and were still living 2 days after the inoculation. Among these, the ones that had been selected for early pupation were smaller (mean wing length 2.68 mm) than the ones selected for late pupation (2.77 mm; F1,4 5 16.9, P , 0.001) with statistically significant differences among lines within selection regime (F4,75 5 3.1, P 5 0.02). Among the 48 inoculated females that had been selected for early pupation, 56% (27) showed no sign of melanization, 38% (18) had patchy melanization and 6% (three) had strongly or completely melanized the bead (Fig. 2). Among the 34 individuals that had been selected for late development, 41% (14) had melanized none of the bead, 26% (nine) had partly and 32% (11) had strongly melanized the bead. Although this difference among selection regimes was significant, neither the difference among lines within selection regime nor the potentially confounding effect of wing length were significant (Table 2).

FIG. 2. Melanization response of mosquitoes to inoculated Sephadex beads as a function of the selection regime. The percentage of mosquitoes that were able to melanize a bead completely, that had an intermediate (i.e., patchy) response, and that were not able to mount a response are shown for each of the six selection lines.

DISCUSSION Selection of mosquitoes for later pupation brought with it larger body size of the mosquitoes and a higher level of immunocompetence, which suggests that the three traits share a genetic basis and are thus genetically correlated. The genetic correlation between age at pupation and immunocompetence will influence the evolutionary pressure on immunocompetence according to standard ideas of life-history theory. An increased level of immunocompetence will generally be advantageous because it increases the adult’s

FIG. 1. Response of age at pupation to selection for early or late pupation. (a) The symbols show the mean age at pupation of each of the mines, the vertical lines the standard errors of the means. Open symbols and dashed lines represent mosquitoes selected for late pupation, closed symbols and solid lines represent mosquitoes selected for early pupation. For clarity, the symbols are slightly shifted along the x-axis. (b) The symbols show the difference of the mean age at pupation between the three early and the three late lines. The vertical lines show the standard error of the difference, calculated according to the equation SE 5 Ïs21/N1 1 s22/N2, where s2i is the variance of line i and Ni is the number of mosquitoes in line i.

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TABLE 2. Ordinal logistic analysis of the melanization response (grouped into three classes) as a function of selection regime. A total of 82 mosquitoes were analyzed. Source

Log-likelihood x2

df

P

Wing length Selection regime Line (selection)

0.412 5.646 2.755

1 1 4

0.521 0.018 0.600

survival rate. Due to the genetic correlation, however, a high level of immunocompetence will also bring with it slow development. Therefore, evolution must find a balance between the advantage of slow development (high adult survival) and its disadvantage (long generation time). This evolutionary conflict is modulated by the genetic correlation between age at pupation and adult size. Such a correlation is quite usual and has been demonstrated previously for A. aegypti (Koella and Offenberg 1999). The genetic correlation shows an additional benefit of delayed pupation, as fecundity generally increases with body size in mosquitoes and in particular in Aedes species (Gilpin and McClelland 1979; Haramis 1985; Packer and Corbet 1989). A similar situation is found for Drosophila melanogaster, in which the immune response against parasitoid infection is associated with low larval competitive ability (Kraaijeveld and Godfray 1997). This cost prevents the spread of resistance in areas where the prevalence of infection is low, whereas in areas with high prevalence the benefit of resistance outweighs its cost, so that the immune system has evolved to kill parasitoid larvae efficiently (Kraaijeveld and Godfray 1999). The association between the correlated responses of body size and melanization response to age at pupation may reflect a re-allocation of resources due to constraints on developmental rates. Mosquitoes that are selected to develop rapidly must use more of their resources for growth than the ones that develop slowly, so that fewer resources are available for immunocompetence. Indeed, it seems clear that developing the immune system requires resources. The melanization response in adult mosquitoes remains weak if they do not obtain a sufficient blood-meal (Chun et al. 1995) or if they have not received sufficient nutrition during their larval development (Suwanchaichinda and Paskewitz 1998). Similar interpretations can be drawn from experiments with other insects. In bumblebees, for example, the encapsulation response is lowest among the most actively foraging insects (Ko¨ nig and Schmid-Hempel 1995). Because flight is the most energetically demanding activity an insect performs (Beenakkers et al. 1984; Casey 1981), this observation can be explained as the consequence of resources being allocated to flight activity and foraging rather than to the maintenance of immunocompetence. This interpretation is strengthened by the more recent findings that mounting an immune response is costly (i.e., increases mortality) only in starved bumblebees (Moret and Schmid-Hempel 2000) and that energetically costly reproductive activity in a damselfly (copulation or oviposition) decreases its encapsulation response (Siva-Jothy et al. 1998). One might further speculate on the role of ecdysone and perhaps other growth hormones in the genetic correlation, as ecdysone not only regulates a wide variety of developmental

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processes in insects, such as molting and metamorphosis, but also the expression of the genes coding for prophenoloxidase (Ahmed et al. 1999). This is a key enzyme in the synthesis of melanin and is involved in the melanization response of mosquitoes. It is also involved in the melanization of eggs (Bodine and Allen 1941) and continues to be expressed throughout the different stages of larval growth (Lee et al. 1998). Thus, the regulation of prophenoloxidase may require developmental decisions between growth and immune function. As Sephadex beads are often used as a model of immunity against malaria parasites (Paskewitz and Riehle 1994; Gorman et al. 1996), our results seem at first in striking contrast to an experiment where A. aegypti mosquitoes were selected for high or low numbers of oocysts (an early developmental stage) of the parasite Plasmodium gallinaceum (Yan et al. 1997). In our study, selection for late pupation increased immunocompetence, whereas resistant mosquitoes in the Yan et al. experiment matured earlier and were smaller than the mosquitoes with high sensitivity. The explanation for this contrast may lie in the fact that resistance against malaria can involve other mechanisms than the encapsulation immune response. Although the mechanisms of resistance are not discussed in Yan et al. (1997), it seems that melanization of oocysts played no role in their experiment. Rather, resistance took place at an earlier stage of the parasite’s development and might have been due to the processes involved in lysis and killing of the parasites within the midgut (Vernick et al. 1995). An additional component of the explanation could be that selection for low oocyst numbers had as a direct result a correlated selection for small mosquitoes because smaller mosquitoes obtain a smaller blood-meal than large ones and are therefore less likely to obtain many oocysts (Lyimo and Koella 1992). In either case, the difference between the two experiments might emphasize the variability of evolutionary patterns for different aspects of the immune response; the encapsulation and melanization response, but perhaps not other aspects of resistance, are positively correlated with age at pupation and body size. The different associations with different aspects of resistance and immunity also beg the question of trade-offs and association between various components of the immune system. Indeed, in bumblebees, for example, there is a negative association between the effectiveness of the melanization response and the antibacterial response (Moret and Schmid-Hempel 2001), although it is not yet known whether this association has a genetic basis. In summary, our results suggest that evolutionary pressure for rapid development and early pupation—and thus for small body size—will lead to adult mosquitoes with weak immunity and that areas differing in parasite pressure should be expected to harbor mosquitoes with different life histories. Slowly developing, large, and thus immunocompetent mosquitoes should be found in areas with intense parasite pressure; more rapidly developing, small mosquitoes should be found in areas with little pressure. The genetic correlation between the immunocompetence expressed as a melanization response and life-history traits, if it is indeed a general correlation of mosquitoes, might also influence ideas about the feasibility of potential malaria control with mosquitoes that are genetically manipulated to be resistant against malaria

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infection. Because the melanization response is one of the ways that mosquitoes can be refractory against Plasmodium species, the genes controlling melanization in Anopheles gambiae are considered to be prime candidates for such genetic manipulation (Collins et al. 1986; Zheng et al. 1997). Naturally, such a program will be successful only if the genes that are responsible for resistance can spread to most of the mosquitoes in a population. If the genetic correlations are general, however, the manipulation of a mosquito’s immunocompetence will bring with it a different life history, making the conditions under which refractory strains can spread more difficult to predict. ACKNOWLEDGMENTS We thank A. Schwartz, C. Bourgouin, J. Clobert, and S. Schjørring for their assistance and discussions; S. Aris-Brosou for providing us with the mosquitoes; and M. Zuk and J. Conner for valuable comments on earlier versions of the manuscript. LITERATURE CITED Ahmed, A., D. Martin, A. G. O. Manetti, S.-J. Han, W.-J. Lee, K. D. Mathiopoulos, H.-M. Mu¨ller, F. C. Kafatos, A. Raikhel, and P. T. Brey. 1999. Genomic structure and ecdysone regulation of the prophenoloxidase 1 gene in the malaria vector Anopheles gambiae. Proc. Natl. Acad. Sci. USA 96:14795–14800. Beenakkers, A. M. T., D. J. Van der Horst, and W. J. A. Van Marrewijk. 1984. Insect flight muscle metabolism. Insect Biochem. 14:243–260. Bell, G., and V. Koufopanou. 1986. The cost of reproduction. Pp. 83–131 in R. Dawkins and M. Ridley, eds. Oxford surveys in evolutionary biology. Vol. 3. Oxford Univ. Press, Oxford, U.K. Bodine, J. H., and T. H. Allen. 1941. Enzymes in ontogenesis (Orthoptera). XV. Some properties of protyrosinases. J. Cell. Comp. Physiol. 18:151–160. Casey, T. M. 1981. Insect flight energetics. Pp. 19–52 in C. F. Herried and C. R. Fourtner, eds. Locomotion and energetics in arthropods. Plenum Press, New York. Christophers, S. R. 1960. Ae¨des aegypti (L.), the yellow fever mosquito: its life history, bionomics and structure. Cambridge Univ. Press, Cambridge, U.K. Chun, J., M. Riehle, and S. M. Paskewitz. 1995. Effect of mosquito age and reproductive status on melanization of sephadex beads in Plasmodium-refractory and -susceptible strains of Anopheles gambiae. J. Invertebr. Pathol. 66:11–17. Collins, F. H., R. K. Sakai, K. D. Vernick, S. Paskewitz, D. C. Seeley, L. H. Miller, W. E. Collins, C. C. Campbell, and R. W. Gwadz. 1986. Genetic selection of a Plasmodium-refractory strain of the malaria vector Anopheles gambiae. Science 234: 607–610. Falconer, D. S. 1989. Introduction to quantitative genetics. Longman, Essex, U.K. Gilpin, M. E., and G. A. McClelland. 1979. Systems analysis of the yellow fever mosquito Aedes aegypti. Fortschr. Zool. 25: 355–388. Gorman, M. J., and S. M. Paskewitz. 1997. A genetic study of a melanization response to Sephadex beads in Plasmodium-refractory and -susceptible strains of Anopheles gambiae. Am. J. Trop. Med. Hyg. 56:446–451. Gorman, M. J., A. J. Cornel, F. W. Collins, and S. M. Paskewitz. 1996. A shared genetic mechanism for melanotic encapsulation of CM-Sephadex beads and a malaria parasite, Plasmodium cynomolgi B, in the mosquito, Anopheles gambiae. Exp. Parasitol. 84:380–386. Gorman, M. J., D. W. Severson, A. J. Cornel, F. H. Collins, and S. M. Paskewitz. 1997. Mapping a quantitative trait locus in-

volved in melanotic encapsulation of foreign bodies in the malaria vector, Anopheles gambiae. Genetics 146:965–971. Haramis, L. D. 1985. Larval nutrition, adult body size, and the biology of Aedes triseriatus. Pp. 431–437 in L. P. Lounibos, J. R. Rey and J. H. Frank, eds. Ecology of mosquitoes: proceedings of a workshop. Florida Medical Entomology Laboratory, Vero Beach, FL. Hoang, A. 2001. Immune response to parasitism reduces resistance of Drosophila melanogaster to desiccation and starvation. Evolution 55:2353–2358. Ilmonen, P., T. Taarna, and D. Hasselquist. 2000. Experimentally activated immune defence in female pied flycatchers results in reduced breeding success. Proc. R. Soc. Lond. B 267:665–670. Klasing, K. C., D. E. Laruin, R. K. Peng, and D. Fry. 1987. Immunologically mediated growth depression in chicks: influence of feed intake, corticosterone and interleukin I. J. Nutr. 117: 1629–1637. Koella, J. C., and J. Offenberg. 1999. Food availability and parasite infection influence the correlated responses of life history traits to selection for age at pupation in the mosquito Aedes aegypti. J. Evol. Biol. 12:760–769. Ko¨nig, C., and P. Schmid-Hempel. 1995. Foraging activity and immunocompetence in workers of the bumble bee, Bombus terrestris L. Proc. R. Soc. Lond. B 260:225–227. Kraaijeveld, A. R., and H. C. J. Godfray. 1997. Trade-off between parasitoid resistance and larval competitive ability in Drosophila melanogaster. Nature 389:278–280. ———. 1999. Geographic patterns in the evolution of resistance and virulence in Drosophila and its parasitoids. Am. Nat. 153: S61–S74. Lee, W. J., A. Ahmed, A. della Torre, A. Kobayashi, M. Asida, and P. T. Brey. 1998. Molecular cloning and chromosomal localization of a prophenoloxidase cDNA from the malaria vector Anopheles gambiae. Insect Mol. Biol. 7:41–50. Lyimo, E. O., and J. C. Koella. 1992. Relationship between body size of adult Anopheles gambiae s.l. and infection with the malaria parasite Plasmodium falciparum. Parasitology 104: 233–237. McKean, K. A., and L. Nunney. 2001. Increased sexual activity reduces male immune function in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 98:7904–7909. Møller, A. P. 1997. Parasitism and evolution of host life history. Pp. 105–127 in D. H. Clayton and J. Moore, eds. Host-parasite evolution: general principles and avian models. Oxford Univ. Press, Oxford, U.K. Moret, Y., and P. Schmid-Hempel. 2000. Survival for immunity: the price of immune system activation for bumblebee workers. Science 290:1166–1168. ———. 2001. Immune defence in bumble-bee offspring. Nature 414:506. Nordling, D., M. Andersson, S. Zohari, and L. Gustafsson. 1998. Reproductive effort reduces specific immune response and parasite resistance. Proc. R. Soc. Lond. B 265:1291–1298. Norris, K., and M. R. Evans. 2000. Ecological immunology: life history trade-offs and immune defense in birds. Behav. Ecol. 11:19–26. Packer, M. J., and P. S. Corbet. 1989. Size variation and reproductive success of female Aedes punctor (Diptera: Culicidae). Ecol. Entomol. 14:297–309. Paskewitz, S., and M. A. Riehle. 1994. Response of Plasmodium refractory and susceptible strains of Anopheles gambiae to inoculated sephadex beads. Dev. Comp. Immunol. 18:369–375. Sheldon, B. C., and S. Verhulst. 1996. Ecological immunology: costly parasite defenses and trade-offs in evolutionary ecology. Trends Ecol. Evol. 11:317–321. Siva-Jothy, M. T., Y. Tsubaki, and R. E. Hooper. 1998. Decreased immune response as a proximate cost of copulation and oviposition in a damselfly. Physiol. Entomol. 23:274–277. Spurlock, M. E., G. R. Frank, and G. M. Willis. 1997. Effect of dietary energy source and immunological challenge on growth performance and immunological variables in growing pigs. J. Anim. Sci. 75:720–726.

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Stearns, S. C. 1992. The evolution of life histories. Oxford Univ. Press, Oxford, U.K. Suwanchaichinda, C., and S. M. Paskewitz. 1998. Effects of larval nutrition, adult body size, and adult temperature on the ability of Anopheles gambiae (Diptera: Culicidae) to melanize beads. J. Med. Entomol. 35:157–161. Vernick, K. D., H. Fujioka, D. C. Seeley, B. Tandler, M. Aikawa, and L. H. Miller. 1995. Plasmodium gallinaceum: a refractory mechanism of ookinete killing in the mosquito, Anopheles gambiae. Exp. Parasitol. 80:583–595. Wagland, B. M., J. W. Steel, R. G. Windon, and J. K. Dineen. 1984. The response of lambs to vaccination and challenge with Trichostrongylus columbriformis: effects of plan of nutrition on, and the inter-relationship between, immunological responsiveness and resistance. Int. J. Parasitol. 14:39–44. Webster, J. P., and M. E. J. Woolhouse. 1999. Cost of resistance:

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relationship between reduced fertility and increased resistance in a snail-schistosome host-parasite system. Proc. R. Soc. Lond. B 266:391–396. Williams, T. D., J. K. Christians, J. J. Aiken, and M. Evanson. 1999. Enhanced immune function does not depress reproductive output. Proc. R. Soc. Lond. B 266:753–757. Yan, G., D. W. Severson, and B. M. Christensen. 1997. Costs and benefits of mosquito refractoriness to malaria parasites: implications for genetic variability of mosquitoes and genetic control of malaria. Evolution 51:441–450. Zheng, L., A. J. Cornel, R. Wang, H. Erfle, H. Voss, W. Ansorge, F. C. Kafatos, and F. H. Collins. 1997. Quantitative trait loci for refractoriness of Anopheles gambiae to Plasmodium cynomolgi B. Science 276:425–428. Corresponding Editor: J. Conner